A Large Population of Small Chloroplasts in Tobacco
Leaf Cells Allows More Effective Chloroplast Movement
Than a Few Enlarged Chloroplasts1
Won Joong Jeong, Youn-Il Park, KyeHong Suh, John A. Raven, Ook Joon Yoo, and Jang Ryol Liu*
Plant Cell Biotechnology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Taejon
305–333, Korea (W.J.J., J.R.L.); Department of Biological Sciences, Korea Advanced Institute of Science &
Technology, Taejon 305–701, Korea (W.J.J., O.J.Y.); Department of Biology, Chungnam National University,
Taejon 305–764, Korea (Y.-I.P.); Department of Biology, Taegu University, Taegu 713–714, Korea (K.S.); and
Division of Environmental and Applied Biology, School of Life Science, University of Dundee, Dundee DD1
4HN, United Kingdom (J.A.R.)
We generated transgenic tobacco (Nicotiana tabacum cv Xanthi) plants that contained only one to three enlarged chloroplasts
per leaf mesophyll cell by introducing NtFtsZ1-2, a cDNA for plastid division. These plants were used to investigate the
advantages of having a large population of small chloroplasts rather than a few enlarged chloroplasts in a leaf mesophyll
cell. Despite the similarities in photosynthetic components and ultrastructure of photosynthetic machinery between
wild-type and transgenic plants, the overall growth of transgenic plants under low- and high-light conditions was retarded.
In wild-type plants, the chloroplasts moved toward the face position under low light and toward the profile position under
high-light conditions. However, chloroplast rearrangement in transgenic plants in response to light conditions was not
evident. In addition, transgenic plant leaves showed greatly diminished changes in leaf transmittance values under both
light conditions, indicating that chloroplast rearrangement was severely retarded. Therefore, under low-light conditions the
incomplete face position of the enlarged chloroplasts results in decreased absorbance of light energy. This, in turn, reduces
plant growth. Under high-light conditions, the amount of absorbed light exceeds the photosynthetic utilization capacity due
to the incomplete profile position of the enlarged chloroplasts, resulting in photodamage to the photosynthetic machinery,
and decreased growth. The presence of a large number of small and/or rapidly moving chloroplasts in the cells of higher
land plants permits more effective chloroplast phototaxis and, hence, allows more efficient utilization of low-incident photon
flux densities. The photosynthetic apparatus is, consequently, protected from damage under high-incident photon flux
densities.
During leaf development, chloroplasts in meristematic cells are differentiated from proplastids,
which are the progenitors of various plastids found
in the root and shoot meristem, embryos, endosperm,
and in young developing leaves. Chloroplasts differentiated from proplastids undergo a secondary set of
divisions that result in a large population of small
chloroplasts in each mesophyll cell. As green photosynthetic plastids, chloroplasts typically measure 5
1
This work was supported by the Ministry of Science and
Technology in Korea (grant no. FGM0040012 to J.R.L.); in part by
the Korea Science and Engineering Foundation (grant through the
Plant Metabolism Research Center to J.R.L.); in part by the Crop
Functional Genomics Center of the 21st Century Frontier Research
Program funded by the Ministry of Science and Technology, Republic of Korea (grant no. CGM0400111 to J.R.L.); in part by the
Korea Science and Engineering Foundation through the Agricultural Plant Stress Research Center at Chonnam National University (to Y.I.P.); and in part by the Korea Science and Engineering
Foundation (grant no. 971– 0511– 059 –2 to K.H.S.).
* Corresponding author; e-mail [email protected]; fax 82–
42– 860 – 4608.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.000588.
112
␮m in diameter, are 1 to 2 ␮m thick, and occupy up
to 70% of the surface area of a cell and approximately 20% of the total cell volume in mature leaf
cells (Ellis and Leech, 1985). In the context of plant
productivity and development of the photosynthetic
surface area, and hence the size and number of leaf
mesophyll cells, it is particularly important to understand what determines the ultimate chloroplast size
and number in leaf cells and how chloroplast division is integrated with mesophyll cell development
(Pyke, 1997).
Chloroplasts divide by a process of binary fission
in which constriction of the envelope membranes
occurs. This process is morphologically and genetically similar to bacterial cell division (Leech, 1976;
Whatley, 1988). Recent genetic approaches for understanding chloroplast division and development using
Arabidopsis clearly indicate a close similarity to the
genetic control of prokaryotic cells. There are two
main lines of research supporting this view. The first
is based on genetic studies using a collection of Arabidopsis mutants with altered sizes and numbers of
chloroplasts per cell, known as arc (accumulation and
replication of chloroplast) mutants (Pyke and Leech,
Plant Physiology,
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Small Chloroplasts and Photosynthesis
1992). The second line of research was provided by
transgenic Arabidopsis with fewer and larger chloroplasts in mesophyll cells, constructed by antisense
suppression/sense expression of AtFtsZ (Osteryoung
et al., 1998; Stokes et al., 2000).
Although considerable progress has been made in
elucidating the molecular mechanisms of plastid binary fission in higher plants, an understanding of
how cells control their plastid number is completely
lacking (Pyke, 1999). A close correlation between the
leaf mesophyll cell size and the number of chloroplasts within the cell clearly indicates that cell size is
a primary determinant of the chloroplast number
(Dean and Leech, 1982). Further, it appears that chloroplast division is initiated only after chloroplasts
have attained a certain size (Ellis et al., 1983). Some
arc mutants of Arabidopsis with greatly enlarged
chloroplasts show continued chloroplast development with normal internal structure (Robertson et al.,
1995, 1996). As a consequence, chloroplast division
appears to be a process independent of chloroplast
development. This implies that the chloroplast division event is an integral part of normal leaf cell
development and that the evolution of higher plants
led to each photosynthetic cell containing many
small chloroplasts rather than a few large ones.
The question as to why the photosynthetic cells of
higher plants contain so many small chloroplasts has
not been addressed by detailed experimentation.
Chloroplast positioning within these cells is controlled over a time period of minutes by the light
supply (Trojan and Gabrys, 1996). Zurzycki (1957)
hypothesized that chloroplast rearrangement as a
function of different incident irradiance values maximizes use of limiting light and minimizes the chance
of photodamage to the photosynthetic apparatus under excess light conditions. Chloroplast movement is
believed to alleviate photodamage to PSII under
high-light conditions (Park et al., 1996) and rapid
rearrangement of chloroplasts can be facilitated by
many small chloroplasts rather than by a few large
ones in each cell. However, we know of no reports
that address the question of chloroplast size in higher
plant cells in relation to the effects of variation in the
light supply at the subcellular, cellular, or wholeplant levels. Therefore, this study was intended to
provide an experimental test of Zurzycki’s hypothesis, which was based on a transgenic tobacco (Nicotiana tabacum cv Xanthi) line with fewer but enlarged
chloroplasts in leaf mesophyll cells. Arabidopsis has
become the definitive system in which to study plant
biology over the last 10 years due to various advantages over other plants with respect to molecular
genetics. However, Arabidopsis has not been widely
utilized in research on photosynthesis, mainly due to
difficulties in analyzing leaf photosynthetic gas exchanges and fractionating subcellular components.
Therefore, we chose to use tobacco plants. Compared
with wild types, NtFtsZ-overexpressed transgenic toPlant Physiol. Vol. 129, 2002
bacco showed normal chloroplast development with
a decreased capacity for chloroplast movement in
response to varying light intensities. The presence of
many small chloroplasts in each cell and/or a greater
capacity for chloroplast phototaxis is probably an
evolutionary adaptation that aids in efficient use of
naturally fluctuating light intensities.
RESULTS
Transgenic Plants with Cells Containing a Few
Enlarged Chloroplasts
Leaves of transgenic tobacco plants in which an
NtFtsZ1-2 was overexpressed have one to three enlarged chloroplasts in each cell throughout development. In contrast, wild-type plants have many small
chloroplasts that increase in number during development. The transgenic phenotypes are consistent with
several arc mutants (Pyke and Leech, 1992) and FtsZ
antisense/sense transgenic plants (Osteryoung et al.,
1998; Stokes et al., 2000) of Arabidopsis.
Growth Characteristics and the
Photosynthetic Apparatus
To characterize the growth performance of three
independent transgenic lines that harbor a few enlarged chloroplasts, T1 and T2 plants were grown
under three different light levels for 10 weeks in a
greenhouse (Table I). Compared with wild-type
plants, all transgenic lines grown under both lowand high-light conditions exhibited retarded growth,
with comparable growth under medium light conditions. Based on plant height, stem thickness, fresh
weight, and size of a fully expanded leaf, transgenic
plants grew more slowly under both limiting and
saturation light levels than wild-type plants (Table I).
However, the Chl and protein contents and the Chl
a/b ratio were similar in transgenic and wild-type
plants. Transgenic plants grown under high-light
conditions had a lower Chl content and a higher Chl
a/b ratio per unit area, suggesting photoinhibitory
damage. These growth characteristics of transgenic
plants under both low- and high-light levels in a
greenhouse were also observed in plants under
growth chamber conditions (data not shown).
Compared with wild-type cells, protoplasts isolated from transgenic plant leaf tissues contain between one and three enlarged chloroplasts per cell
(Fig. 1, A and B). These transgenic plant chloroplasts
are much larger than wild-type plant chloroplasts
and have variable shapes (Fig. 1, C and D). This low
number of chloroplasts per cell is maintained
throughout leaf development. Electron micrographs
of chloroplasts from wild-type and transgenic plants
grown under high-light conditions were examined.
Both types of chloroplasts showed thylakoids differentiated into grana and non-stacked membrane
regions with a similar extent of grana membrane
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113
Jeong et al.
Table I. Plant growth characteristics
PH, Plant height (cm). ST, Stem thickness (cm). LA, The youngest fully expanded leaf size (estimated by leaf area, cm2). FW, Fresh wt (g⫺1 leaf
of the youngest fully expanded leaf. Chl a/b, Chlorophyll a/b ratio. Chl, Chlorophyll content (mmol m⫺2). Protein, The soluble protein content
(g m⫺2). Chl a/b, Chl and protein were measured on the fully expanded leaves, third to fifth from the apex of the plants. WT, Wild-type plants.
FtsZ, Average of three independent FtsZ1-2 overexpressed transgenic tobacco plants. GUS, GUS-expressed transgenic tobacco plants. HL, High
light (700 –1,500 ␮mol photons m⫺2 s⫺1). ML, Medium light (200 – 400 ␮mol photons m⫺2 s⫺1). LL, Low light (30 –150 ␮mol photons m⫺2 s⫺1).
Mean values (⫾SE) for eight to 15 plants are shown.
Treatment
HL
WT
FtsZ
GUS
ML
WT
FtsZ
GUS
LL
WT
FtsZ
GUS
PH
ST
LA
FW
Chl
Chl a/b
Protein
69.37 ⫾ 3.81
36.56 ⫾ 6.65
60.12 ⫾ 5.54
1.56 ⫾ 0.09
1.22 ⫾ 0.15
1.57 ⫾ 0.15
499.13 ⫾ 71.29
341.22 ⫾ 89.74
516.17 ⫾ 50.95
13.41 ⫾ 1.91
9.09 ⫾ 2.54
13.87 ⫾ 1.36
0.37 ⫾ 0.030
0.27 ⫾ 0.006
0.36 ⫾ 0.003
3.70 ⫾ 0.13
4.22 ⫾ 0.12
3.65 ⫾ 0.14
1.55 ⫾ 0.10
1.54 ⫾ 0.16
1.49 ⫾ 0.20
48.23 ⫾ 5.01
40.24 ⫾ 3.23
47.40 ⫾ 3.50
0.85 ⫾ 0.09
0.80 ⫾ 0.06
0.86 ⫾ 0.07
200.75 ⫾ 47.02
186.88 ⫾ 33.72
218.44 ⫾ 55.08
4.89 ⫾ 1.14
4.53 ⫾ 0.82
5.32 ⫾ 1.34
0.31 ⫾ 0.010
0.30 ⫾ 0.009
0.33 ⫾ 0.011
3.30 ⫾ 0.05
3.47 ⫾ 0.01
3.43 ⫾ 0.02
1.23 ⫾ 0.02
1.40 ⫾ 0.09
1.19 ⫾ 0.01
29.20 ⫾ 5.05
8.80 ⫾ 3.14
26.62 ⫾ 9.63
0.47 ⫹ 0.09
0.28 ⫾ 0.06
0.48 ⫾ 0.08
110.21 ⫾ 17.73
42.27 ⫾ 14.22
92.10 ⫾ 20.42
2.47 ⫾ 0.39
0.92 ⫾ 0.33
2.07 ⫾ 0.45
0.20 ⫾ 0.005
0.19 ⫾ 0.005
0.20 ⫾ 0.004
2.91 ⫾ 0.02
2.98 ⫾ 0.05
2.99 ⫾ 0.01
0.48 ⫾ 0.04
0.48 ⫾ 0.05
0.52 ⫾ 0.05
stacking (Fig. 1, E and F). Unlike the wild-type chloroplasts, highly extended bundles of microtubulelike structures in the stroma were observed in
electron micrographs of transgenic chloroplasts (data
not shown).
The contents of the PSII reaction center D2
polypeptide Cytf, the PSI reaction center heterodimer
(PsaA/PsaB), and light-harvesting pigments Lhcb1
and Lhcb2 were examined on a Chl basis using immunoblotting (Fig. 2). The relative Rubisco content
also was determined spectrophotometrically on a soluble protein basis (data not shown). The photosynthetic components of transgenic plants that were
examined showed no significant changes compared
with the wild type.
Chloroplast Movements and Photosynthesis
To investigate differences in chloroplast rearrangement between the two plant types, chloroplast movements were determined microscopically and by
changes in the amount of light transmitted through
the leaf tissue. Light micrographs of wild-type and
transgenic leaf sections were examined before and
after illumination treatments. High-light treatment
induced significant changes in chloroplast arrangement in wild-type plants relative to transgenic plants
(Fig. 3). Wild-type chloroplasts in leaves pretreated
in the dark were mainly located along walls perpendicular to the light direction (face position). Exposure
to high photon flux densities induced chloroplast
movement from the face position to the profile position where chloroplasts were aligned with the cell
walls parallel to the light direction. In contrast, rearrangement of transgenic tobacco chloroplasts in response to light conditions was not evident (Fig. 3, C
and D).
Transmittance changes have been described by the
sieve effect and provide a convenient and nonde114
structive method for monitoring chloroplast movement. Hence, chloroplast rearrangement in transgenic plants was also investigated by leaf transmittance changes. Leaves maintained in the dark for
20 min were first illuminated with low light (40 ␮mol
m⫺2 s⫺1) for 30 min, followed by high light (750 ␮mol
m⫺2 s⫺1) for 30 min (Fig. 4A). Leaf transmittance in
wild-type tissues decreased under low light, then
increased rapidly under high-light illumination.
Changes in transmittance under both irradiances
were completed within 30 min, resulting in changes
in leaf absorbance estimated either at 660 nm or in
the 400- to 700-nm range (Table II). Contrary to the
typical traces for changes in leaf transmittance, transgenic leaves showed greatly diminished changes in
leaf transmittance under both irradiance levels.
These changes in leaf transmittance are indicative of
chloroplast movement because leaf tissues of both
types treated with the actin antagonist cytochalasin D
did not show any significant changes in transmittance. Therefore, the diminished changes in leaf
transmittance observed in transgenic plant tissue
strongly suggest that chloroplast rearrangement is
severely retarded, consistent with the results shown
in Figure 3.
Photosynthetic induction responses based on leaf
CO2 exchange rates under limiting and saturating
irradiances at a saturating CO2 pressure (850 ␮L L⫺1)
were simultaneously measured with leaf transmittance values (Fig. 4, B and D). Upon illumination of
wild-type plant leaves under low light (40 ␮mol m⫺2
s⫺1) following a 10-min dark period, there was a
slight rise of the photosynthetic CO2 uptake rate,
followed by a lag period and a gradual increase to
steady-state conditions with a half maximum rate
after 10 min of illumination. Compared with the
photosynthetic induction curve under low-light conditions, illumination of leaves under saturating light
following low light showed similar photosynthetic
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Plant Physiol. Vol. 129, 2002
Small Chloroplasts and Photosynthesis
Figure 2. Representative immunoblots of the light harvesting Chl
pigments of the two photosystems (Lhca1 and Lhca2), the PSII reaction center D2 protein, the PSI reaction center PsaA/B heterodimer,
and Cytochrome f (Cyt f) from isolated thylakoid membranes.
Figure 1. Light (A and B), confocal (C and D), and ultramicroscopic
(E and F) photomicrographs of protoplasts, chloroplasts, and internal
chloroplast structures from wild-type (A, C, and E) and transgenic (B,
D, and F) tobacco. The bars in A through D and E through F are 10
and 1 ␮m, respectively.
lincomycin-treated tobacco leaf discs were floated on
water and illuminated at 1,600 ␮mol m⫺2s⫺1 for various periods up to 6 h. Photoinhibition was assayed
by the Chl fluorescence ratio (Fv/Fm), a measure of
the quantum efficiency of PSII photochemistry (Fig.
5B). Lincomycin was used to prevent replacement of
damaged D1 proteins during light treatment and
allowed us to assess the gross photoinhibition of PSII.
With an increasing treatment time, there was an
abrupt decline in the Fv/Fm ratio during the first 2 h
of light treatment, followed by a phase of constant
low values. Clearly, the Fv/Fm ratio of wild-type
induction kinetics without any significant lag period.
Transgenic plants showed three-phase induction kinetics with an extended lag period, and steady-state
photosynthetic rates similar to the wild type (Fig. 4B).
Measurements of photosynthetic rates (Pn) at a
saturating light were supplemented by experiments
at CO2 levels equal to, above (700 and 500 ␮L L⫺1),
and below (200, 125 and 50 ␮L L⫺1) the present
atmospheric CO2 value of approximately 360 ␮L L⫺1
in postinduction light regimes. Results for net photosynthetic CO2 exchange rates, Pn as a function of
the intercellular space CO2 (Ci) for a number of wild
and transgenic lines (Fig. 4C), showing no significant
differences in the photosynthetic carbon assimilation
between wild and transgenic plants.
Photoinhibition of Photosynthesis
Differences in susceptibility to high-light conditions between wild-type and transgenic tobacco
plants grown under low-light conditions were measured. Control (distilled water [DW] treated) or
Plant Physiol. Vol. 129, 2002
Figure 3. Comparison of light photomicrographs of cross sections of
tobacco leaves, wild-type (A and B), and transgenic (C and D) plants.
Leaf sections were taken before (A and C) and after (B and D)
illumination under high light (750 ␮mol m⫺2 s⫺1) for 30 min. The bar
represents 100 ␮m.
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115
Jeong et al.
Figure 4. Changes in leaf transmittance (A) and CO2 exchange rates (B) in tobacco leaves measured at 660 nm. Darkadapted intact leaves were first illuminated under low light (40 ␮mol m⫺2 s⫺1), then immediately followed by high light (750
␮mol m⫺2 s⫺1). WT-Cont, Wild type treated with DW; FtsZ-Cont, transgenic plant treated with DW; WT & FtsZ-CD,
wild-type and transgenic plants treated with 5 ␮M cytochalasin D; wild type (f and F); transgenic plant (䡺 and E). C, Net
photosynthesis (Pn) versus intracellular CO2 concentration (Ci) in attached leaves of the wild-type (F) and transgenic (E)
tobacco plants. The light intensity for CO2 exchange was 750 ␮mol m⫺2 s⫺1 and leaf temperature was 25°C ⫾ 1°C. D,
Schematic diagram of a Parkinson leaf chamber modified for the simultaneous measurement of optical properties and gas
exchange. Two optic fibers were inserted into the chamber to collect lights reflected directly from an intact sample and from
a small block coated with barium sulfate mounted on the bottom of the chamber, respectively.
plants was higher than transgenic plants throughout
the experimental period, showing that the PSII reaction centers of transgenic plants are more susceptible
to photoinhibition. Compared with the control, lincomycin treatment accelerated photoinhibition of
PSII, especially in wild-type plants. However, the
PSII units of transgenic plants were more susceptible
to photoinhibitory light treatment than wild-type
plants. As expected, plants grown under high-light
conditions in a greenhouse often showed the photoinhibitory symptom of photobleaching at the leaf
blade (Fig. 5A). When light response curves of photosynthetic O2 evolution were measured using plants
that did not show any visible symptoms, as shown in
Figure 5A, the photosynthetic rate of transgenic plants
was approximately 40% lower than wild-type plants
116
under all light regimes (Fig. 5C), clearly indicating that
the photosynthetic machinery of the transgenic plants
is more susceptible to photodamage.
DISCUSSION
Why higher plant chloroplasts divide rather than
simply expand is unknown. Two different types of
plants, one with many small chloroplasts and the
other with a few enlarged chloroplasts per leaf mesophyll cell, were systematically compared under
varying levels of incident light. Transgenic tobacco
plants with a few enlarged chloroplasts per cell were
generated by overexpression of the NtFtsZ gene. The
overall growth of transgenic plants was retarded under both low- and high-light conditions and was
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Plant Physiol. Vol. 129, 2002
Small Chloroplasts and Photosynthesis
comparable under medium-light conditions (Table I).
However, Arabidopsis FtsZ transgenic plants and arc
mutants with a few enlarged chloroplasts per cell are
not distinguishable from wild-type plants in outward
appearance and growth (Pyke et al., 1994; Robertson
et al., 1995, 1996; Osteryoung et al., 1998). In this
respect, transgenic tobacco plants are different from
the arc mutants and transgenic plants of Arabidopsis.
Considering the observed plant productivity and leaf
mesophyll functionality in photosynthesis, these differences in growth patterns imply that cells with
many small and/or rapidly moving chloroplasts derive certain benefits in absorption and utilization of
light energy that are not available to cells with a few
gigantic and/or slowly moving chloroplasts. Plant
growth ultimately depends on leaf photosynthesis,
which is determined by the rate of excitation of the
photosystem and utilization of this excitation energy
through electron transport and metabolism, as constrained by the supply of carbon dioxide to Rubisco
(Huner et al., 1998). Therefore, although there are
differences in assimilation allocation among organs
between wild-type and transgenic plants, some of the
differences in plant growth arise from changes in
either the photosynthetic structure or the functional
performance of the photosynthetic apparatus
(Slatyer, 1970). To clarify this point, we first examined the ultrastructure of the chloroplast and the
relative stoichiometry of various photosynthetic
components (Figs. 1 and 2). The ultrastructure of the
chloroplast, including the degree of grana stacking
that is involved in adjustment of the photosynthetic
apparatus to a fluctuating light environment, is common to both wild-type and transgenic plants. Further, quantification of the photosynthetic components involved in light energy absorption and
electron transport, carbon dioxide fixation, and carbon assimilation rates clearly indicates that all of the
photosynthetic components measured are similar in
the two plant types. All ultrastructural, biochemical,
and gas exchange data indicate strongly against a
growth difference being due to a change in the composition of the chloroplasts induced by inhibiting
chloroplast division.
Despite similar photosynthetic structures and functions at the subchloroplast level, differences were
observed at the whole organism level. The growth
performance of transgenic plants with a few enlarged
chloroplasts was inferior to plants with many small
chloroplasts. To determine whether inefficient light
absorption is responsible for this observed difference
in growth performance, we measured the physical
antenna size of the two photosystems and the rearrangement ability of the chloroplasts. The rate of
light absorption per reaction center is determined by
the physical antenna size of each photosystem (Mauzerall and Greenbaum, 1989; Huner et al., 1998) and
the arrangement of chloroplasts (Haupt and Scheuerlein, 1990; Augustynowicz and Gabrys, 1999). The
Plant Physiol. Vol. 129, 2002
Chl contents, the Chl a/b ratio, and western-blot data
for Lhca1 and Lhcb1, which are parameters often
used to describe the physical antenna size, were comparable between the two types (Table I; Fig. 2). However, dramatic differences in chloroplast rearrangement and leaf optical properties between wild-type
and transgenic plants were observed (Figs. 3 and 4A;
Table II). This indicates involvement of chloroplast
movement in the observed differences in plant
growth. Decreased growth is probably due to decreased light absorption (limiting conditions) and an
increased susceptibility to photoinhibition (highlight conditions). Under limiting light, photosynthesis is often limited by the rate of light absorption,
which is, in turn, dependent on the physical antenna
size. However, the fact that the physical antenna
sizes are comparable between the two types of plants
indicates that the observed differences in growth performance under low-light conditions result from leaf
optical changes at the whole plant level, not at the
leaf level.
In productive agricultural ecosystems, the values
of the leaf area index and the ratio of photosynthetic
leaf area to covered ground area fall in the range of 3
to 5 (Hopkins, 1999). This means that utilization of
light by leaves in the canopy is an important factor in
plant productivity. At the whole plant level, a lowered transmittance in the outermost leaves of transgenic plants might result in a limitation of light available for photosynthesis to inner leaves in the canopy,
especially when the light supply is the limiting factor
for photosynthesis in these leaves. Because the availability of light for plants growing under lightlimiting conditions is the most important factor determining photosynthetic activity, a lowered transmittance results in decreased primary productivity.
In addition, the outermost leaves of transgenic plants
exposed to varying light intensities would be subject
to an increased potential for photoinhibition. This
view is partly supported by the greater susceptibility
of photosystem II of transgenic plants to high light
compared with wild-type plants, and the lowered
photosynthetic O2 evolution rate under all light regimes (Fig. 5). Thus, the observed poor growth of
transgenic plants may be attributable to reduced photon absorption by shaded leaves and a greater tendency for photoinhibition in the outermost leaves.
However, it must be borne in mind that allocation of
photosynthate to production of new photosynthetic
machinery (leaf area), or to other sinks in the plant, is
also a determining factor for how photosynthesis on
a leaf area basis relates to the plant growth rate
(Slatyer, 1970). Besides the allocation of photosynthate, we do not exclude the possibility that poor
growth can be related to a reduced surface area to
volume ratio of enlarged chloroplasts, and affected
proplastid division in growing zones (Robertson et
al., 1995). However, in this study, growth performance of transgenic tobacco plants grown under
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Jeong et al.
Table II. Leaf optical properties
WT, Wild-type plants. FtsZ, FtsZ1-2-overexpressed transgenic tobacco plants. Leaf transmittance, reflectance, and absorptance were measured
from leaves of plants grown under medium-light conditions. Leaf transmittance and reflectance measured at 660 nm in the visible light range
(400 –700 nm, mean of the 92 measurements between 399.89 and 700.05 nm, each one-half band width ⫽ about 2.5 nm) were measured after
30 min of illumination under low light (LL; 40 ␮mol m⫺2 s⫺1) and high light (HL; 750 ␮mol m⫺2 s⫺1), respectively. Mean values (⫾SE) for four
to six leaves are shown.
660 nm
400 –700 nm
Optical Property
Transmittance
LL
HL
Reflectance
LL
HL
Absorptance
LL
HL
WT
FtsZ
WT
FtsZ
0.094 ⫾ 0.007
0.120 ⫾ 0.014
0.105 ⫾ 0.0089
0.106 ⫾ 0.0084
0.029 ⫾ 0.003
0.066 ⫾ 0.014
0.040 ⫾ 0.006
0.049 ⫾ 0.008
0.120 ⫾ 0.001
0.130 ⫾ 0.001
0.154 ⫾ 0.011
0.143 ⫾ 0.012
0.090 ⫾ 0.004
0.088 ⫾ 0.003
0.107 ⫾ 0.007
0.098 ⫾ 0.008
0.778 ⫾ 0.006
0.749 ⫾ 0.014
0.742 ⫾ 0.019
0.751 ⫾ 0.019
0.881 ⫾ 0.003
0.846 ⫾ 0.012
0.853 ⫾ 0.012
0.853 ⫾ 0.015
medium-light conditions was comparable with wildtype plants, eliminating the possibility of impaired
growth due to a decreased surface area to volume
ratio.
The simplest assumption is that the cellular level
responses of photosynthesis related to differences in
chloroplast movement and distribution are the major
Figure 5. A, Photographs showing photoinhibitory damage in leaves
of transgenic (FtsZ) tobacco. B, Photoinhibition of photosystem II
estimated by the Chl fluorescence ratio Fv/Fm as a function of the
high-light illumination period in leaf discs of wild-type (f and F) and
transgenic (䡺 and E) plants grown for 10 weeks under low light.
Lincomycin (F and E) treatment was applied for inhibition of the
photodamaged PSII reaction center. C, Light response curve of photosynthetic O2 evolution in leaf discs of the wild-type (f) and transgenic (䡺) tobacco plants grown under high-light conditions. O2
evolution in leaf discs were measured at 5% (v/v) CO2 concentration
at 25°C ⫾ 1°C. Mean values (⫾SE) for 15 leaf discs are shown.
118
causes of the slower growth of transgenic plants.
These observations are consistent with reports of various species that exhibit chloroplast movement, supporting Zurzycki’s hypothesis of a dual function for
chloroplast movement in ensuring maximum light
absorption under limiting light and protecting chloroplasts from photodamage (Zurzycki, 1957).
In interpreting the effects of restricting FtsZ expression in tobacco, we can rely on comparative biology,
looking to multicellular organisms that naturally
have only one chloroplast per cell compared with
organisms containing many chloroplasts in each cell.
Particular reference is made to the speed at which
chloroplasts respond phototactically and the extent
to which a single large chloroplast can restrict the
inorganic carbon supply to Rubisco, relative to many
small chloroplasts per cell.
Examples of organisms from the embryophytes
and the closely related charophycean algae that naturally have only one plastid per cell are the algae
Coleochaete pulvinata, Klebsormidium flaccidum (formerly Hormidium), and Mougeotia sanfordiana (Van
den Hoek et al., 1995), the anthocerote bryophytes
(hornworts), and some leaf cells in Selaginella spp. Of
these, Klebsomormidium, Mougeottia, and Selaginella
show phototactic plastid movements, whereas the
time necessary for the phototactic repositioning of
the plastid to occur may be somewhat longer than in
embryophyte cells with many small plastids (Haupt
and Scheuerlein, 1990; Augustynowicz and Gabrys,
1999). The difference is much less than that between
wild-type and NtFtsZ-overexpressed tobacco plants,
indicating that the few, large, slowly responding
chloroplasts of the transgenic plants probably have
restricted phototactic movement for a reason other
than their large size.
The data presented here and reported earlier
(Chow, 1994; Park et al., 1996) indicate that chloroplast movement is an effective photoprotection
mechanism in land plants in both exposed (tobacco)
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Plant Physiol. Vol. 129, 2002
Small Chloroplasts and Photosynthesis
and shaded forest floor (Tradescantia albiflora) habitats. Large-scale nastic movements can also serve in
photoprotection, e.g. in the leaflet folding of forest
floor species of Oxalis (Raven, 1989). It is possible that
the stem movements and leaf rolling of some Selaginella spp. that grow exposed to full sun in the dry
summer are photoprotective responses. Movement
and rolling are absent in the rainy winter season
when there is less light (e.g. Selaginella tamariscina). It
is not yet clear, however, whether the bending and
rolling is mainly a response to excess light or insufficient water. The involvement of phototactic chloroplast movement in photoprotection is also found in
benthic aquatic plants.
Some algae (e.g. M. sanfordiana) with a single
plank-like plastid in each have parts of the plastid at
least 10 ␮m from the plasmalemma; however, this
may not impact on inorganic carbon supply to
Rubisco because these algae, unlike tobacco, have an
inorganic carbon concentrating mechanism. Any
problems with CO2 diffusion in transgenic tobacco
for increased FtsZ expression can be related to the
larger radial dimension of a few enlarged chloroplasts, and especially to the large distance between
the plasmalemma and the chloroplast envelope.
In conclusion, based on transgenic tobacco plants
with a few enlarged chloroplasts, we suggest that
natural selection pressure favors cells with many
small chloroplasts over those with a few enlarged
chloroplasts by efficient utilization of radiant energy
and minimization of photodamage under varying
light conditions, as suggested by Zurzycki (1957).
MATERIALS AND METHODS
Transgenic Tobacco (Nicotiana tabacum cv Xanthi) and
Plant Growth
NtFtsZ1-2 (FtsZ1-2 of tobacco) was overexpressed in tobacco under control of the cauliflower mosaic virus promoter. Three independent transgenic tobacco plants were
confirmed by Southern, northern, and western blotting
(data not shown). Northern- and western-blot analyses
showed that FtsZ1-2 and FtsZ1-2 in transgenic plants were
overexpressed about 3 to 12 times more than wild type.
Transgenic plants consisted of two distinct phenotype
classes. The first had a few enlarged chloroplasts and the
second had one gigantic chloroplast with several normalsized chloroplasts in mature cells. Plant growth comparisons were based on the T1 and T2 progeny of three independent transgenic plants with one to three enlarged
chloroplasts, wild-type plants, and transgenic plants expressing GUS (a parallel control) grown for 10 weeks in a
greenhouse. Typical temperatures reached 32°C and irradiance during the day ranged from 750 to 1,500 ␮mol m⫺2
s⫺1. The maximum irradiance during the day was defined
arbitrarily as 100% (high light), 20% (medium light), and
5% (low light). Relative growth irradiances were obtained
using layers of shade cloth. T1 plants containing between
one and three greatly enlarged chloroplasts were selected
Plant Physiol. Vol. 129, 2002
under a light microscope and the fully expanded leaves,
third to fifth position from the apex, of the T1 plants used
for further experiments.
Inhibitor Treatments
Leaf petioles were cut under water and excised leaves
were transferred to 10-mL Falcone tubes containing either
5 ␮m cytochalasin D (actin antagonist; Sigma, St. Louis) or
1 mg mL⫺1 lincomycin (protein synthesis inhibitor; Sigma)
for 10 h under dim light (10 ␮mol m⫺2 s⫺1).
Light, Confocal, and Electron Microscopy
Light and confocal micrographs were measured on protoplasts from the leaves from 6-week-old plants in the
growth chamber. Electron micrographs of chloroplasts
from wild-type and transgenic plants grown for 10 weeks
under high light in the greenhouse were examined. Light
microscopic observations for the chloroplast movement
were taken from tissue pieces approximately 1 mm long
and 0.5 mm thick from leaves grown for 10 weeks under
the medium-light greenhouse condition. Before high-light
(750 ␮mol m⫺2 s⫺1) treatment for 30 min, leaves were fed
either with 5 ␮m cytochalasin D or distilled water for about
10 h under dim light through cut petioles. Leaf sections
were fixed immediately in ice-chilled 3% (v/v) glutaraldehyde for 1 h under high light to prevent chloroplast rearrangement during fixation (Park et al., 1996). Leaf sections
were stained with toluidine blue, then observed and photographed. For confocal microscopic observations, leaf protoplasts were isolated by incubation from leaves and
placed in an enzyme digestion solution (2% [w/v] cellulase
Onozuka R-10, 1% [w/v] Macerase R-10, 0.6 m mannitol,
and filter-sterilized 5 mm MES) overnight. Protoplasts purified by filtering through a mesh, centrifugation, and
washing, and were embedded in 0.6% (w/v) agarose. Images of Chl autofluorescence were observed with an LSM
410 confocal microscope (Zeiss, Overkochen, Germany) and
photographed. For electron microscopic observations, leaf
tissues were fixed in phosphate-buffered 3% (v/v) glutaraldehyde, followed by 1% (w/v) OsO4, dehydrated, and embedded in Spurr’s resin. Thin sections were stained with
lead citrate and examined with a Zeiss EM 912 Omega at
80 kV.
Determination of the Photosynthetic Components
Relative leaf Rubisco contents from wild-type and transgenic tobacco plants were determined spectrometrically
after formamide extraction of Coomassie Brilliant Blue
R-250-stained subunit bands separated by SDS-PAGE
(Makino et al., 1997). Soluble protein contents were measured using protein assay reagents according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules,
CA). Thylakoidal protein components were measured immunochemically after isolation of thylakoid membrane
proteins. Thylakoid proteins were prepared from protoplasts using methods modified from Gray et al. (1996).
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119
Jeong et al.
Thylakoid membranes were isolated from the protoplasts
of the fully expanded leaves, third to fifth position from the
apex, of the plants grown for 10 weeks under the mediumlight condition in the greenhouse. Protoplasts were broken
in 10 mm sodium phosphate buffer containing 5 mm NaCl
and 5 mm MgCl2. Thylakoid membranes were resuspended
in 10 mm Tricine-NaOH, pH 7.0; 300 mm Suc; and 5 mm
MgCl2. Chl contents were determined in 80% (v/v) acetone
by the method of Porra et al. (1989). For protein gel blots,
membranes were solubilized in 60 mm Tris-HCl (pH 7.8),
12% (w/v) Suc, 2% (w/v) SDS, 1 mm EDTA, and 58 mm
dithiothreitol. Protein gel electrophoresis was performed
according to Laemmli (1970). Separated proteins were electrophoretically transferred to Immobilon-P (Millipore, Bedford, MA). Immunodetection by chemiluminescence using
antibodies was performed according to the manufacturer’s
instructions (ECL ⫹ Plus; Amersham-Pharmacia Biotech,
Uppsala). Antibodies against Lhca1 and Lhcb1, Psa A/B,
D2, and Cytf were provided by Drs. Stefan Jansson (Umea,
Sweden), Kintake Sonoka (Tokyo), MinKyun Kim (Suwon,
Korea), and Amane Makino (Tohoku, Japan), respectively.
Electronics, Seoul) and a temperature controller (MX7,
Hanyong Co., Seoul).
Determination of Chl Fluorescence Parameters and
Photosynthetic O2 Evolution
The maximum efficiency of PSII was estimated from the
Chl fluorescence ratio Fv/Fm at room temperature. After
photoinhibitory light treatments at 1,600 ␮mol m⫺2 s⫺1 for
varying durations, leaf discs from plants grown for 10
weeks under low-light conditions in a growth chamber
were dark treated for 30 min in leaf clips of a Plant Efficiency Analyzer (Hansatech, King’s Lynn, UK). Lightresponse curves of photosynthetic O2 evolution during
illumination were determined with a leaf disc O2 electrode
(Oxygraph system, Hansatech) using plants grown under
high-light conditions in a greenhouse. Various irradiances
were provided using neutral density filters while the temperature was kept constant at 25°C at 5% (v/v) CO2.
ACKNOWLEDGMENTS
Leaf Gas Exchange and Optical Property
A Parkinson leaf chamber (broad type, ADC, Hoddesdon, Hertfordshire, UK) was modified for simultaneous
measurements of the CO2 exchange rate and of the transmittance and reflectance in the range of 400 to 700 nm (Fig.
4D) for sample leaves from 10-week-old plants under the
medium-light condition in the greenhouse. One of two
optic fibers was inserted through the upper lid of the
chamber at an angle of 60° to the plane of the lid to collect
and guide the light reflected directly from the upper surface of the leaf. The other optic fiber was inserted at right
angles through the lower lid of the chamber to collect and
guide the light that had traversed the leaf and then been
diffused from a small triangular cross section block coated
with barium sulfate located at the bottom of the chamber
with the face at 45° to the leaf surface. Transmittance was
determined by dividing the photon flux density detected
by the lower probe with the leaf in place by that detected in
the absence of the leaf. Reflectance was measured by dividing the photon flux density detected by the upper probe
with the leaf in place by that measured when the leaf was
replaced by a 1-mm-thick plastic panel coated with barium
sulfate. The spectral intensity of the light collected and
guided by the optic fibers was measured with a silicon
photodiode detector (MMS, Zeiss). CO2 exchange rates at
saturating CO2 (850 ␮L L⫺1) were measured with a steadystate gas exchange system (LCA2, ADC). Incident photon
flux densities (40 and 750 ␮mol m⫺2 s⫺1) from a halogen
lamp (12W DC, Philips, Eindhoven, The Netherlands) were
provided with neutral density filters. CO2 exchange rates
for Pn verus Ci curve were measured at a saturating light
intensity (750 ␮mol m⫺2 s⫺1). Air containing various concentrations of CO2 (50, 125, 200, 360, 500, and 850 ␮L L⫺1)
and preconditioned to 40% relative humidity was supplied
to the chamber at 250 mL min⫺1. The leaf temperature was
maintained at 25 ⫾ 1°C with a Peltier cooler (Sungjoo
120
We thank Dr. Wah Soon Chow for critical review of the
manuscript. We also thank the Korea Basic Science Institute
for preparation of microscopic images.
Received November 16, 2001; returned for revision December 13, 2001; accepted February 7, 2002.
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